Intrinsically fluorescent cytotoxic cisplatin analogues as DNA marker molecules

Two square planar derivatives of Pt(en)Cl(2) with intrinsic fluorescence in aqueous solution at room temperature, with quantum yields (Phi) 0.11 and 0.10, respectively, have been synthesized and characterized as [Pt(en)(CG)Cl] (Complex 1) and [Pt(en)(CG)(2)] (Complex 2) (en = ethylenediamine, CG = cholylglycinate). Complexes 1 and 2 exchange just one ligand (chloride or cholylglycinate, respectively) when reacted with water or 5'-GMP to give the same chemical species. After reaction with DNA oligonucleotides or DNA plasmids, they show enhanced emission in the visible region, which lasts for long periods of time and makes them potentially useful DNA marker molecules. Incubation with nucleated blood cells followed by microscopic analyses revealed that they enter the cells within minutes of exposure, selectively stain the DNA, and persist after more than 48 h of exposure. Complexes 1 and 2 display cell cycle phase-independent cytotoxic activity against cisplatin-resistant CHO (Chinese hamster ovarian) tumor cells, with an early onset of their effects. Their slightly different biological effects, as compared to cisplatin, are considered to be linked to the bile acids and their vector properties and to the preferential formation of monoadducts.     

Treatment of genotype 1b HCV-related chronic hepatitis: efficacy and toxicity of three different interferon alfa-2b/ribavirin combined regimens in naive patients

This prospective open-label randomized trial of chronic hepatitis C genotype-1b patients compared compared the efficacy and safety of peg-interferon alfa-2b administered once-weekly versus interferon alfa-2b thrice-weekly or daily, both in combination with ribavirin. Seventy-eight previously untreated patients, with biopsy-documented genotype 1 chronic HCV and persistently elevated ALT levels and detectable HCV RNA, were randomized (26 subjects each) to receive: interferon alfa-2b at 6MIUs.c./three-times-weekly (group A) or interferon alfa-2b, 3MIUs.c./daily (group B) or peg-interferon alfa-2b 1.5mcg/Kg s.c./once-weekly (group C). All regimens included standard weight-based doses of ribavirin (800, 1,000 or 1,200 mg/day) administered for 52-weeks. Patients in the three groups were comparable for age, sex, viral load, ALT value and histological-activity-index (HAI). Therapy was completed by 22, 20 and 23 patients in groups A, B and C, respectively. At the end of treatment, a complete (biochemical and virological) response was observed in 50.0% patients of group A, 57.7% of group B and 65.4% of group C. After an additional 24-weeks of follow-up, a sustained response was observed in 26.9%, 46.1% and 50.0% of patients in groups A, B or C, respectively. Therapy was discontinued by 4, 6 and 2 patients because of adverse events in the above three groups. In naive patients with chronic genotype-lb hepatitis C, a 48 week therapy with peg-interferon or interferon at daily doses combined with ribavirin were both more effective than treatment with thrice-weekly interferon in inducing end of treatment and sustained response. Peg-interferon treatment was better tolerated and provoked significantly fewer therapy discontinuations.     

Multiple recurrences of vertebral hydatidosis: a case report

We present a case of an Italian man with a particularly prolonged history of hydatid disease (more than 20 years, with six recurrences) involving both osseus (costal and vertebral) and visceral (pulmonary) sites. The main clinical problems involved in the diagnosis and management of vertebral manifestations of hydatidosis are discussed in the light of the latest clinical research.     

Efficient preservation in a silicon oxide matrix of Escherichia coli, producer of recombinant proteins

The aim of this work was to study the use of silicon oxide matrices for the immobilization and preservation of recombinant-protein-producing bacteria. We immobilized Escherichia coli BL21 transformants containing different expression plasmids. One contained DNA coding for a T-cell receptor β chain, which was expressed as inclusion bodies in the cytoplasm. The other two encoded bacterial superantigens Staphylococcal Enterotoxin G and Streptococcal Superantigen, which were expressed as soluble proteins in the periplasm. The properties of immobilization and storage stability in inorganic matrices prepared from two precursors, silicon dioxide and tetraethoxysilane, were studied. Immobilized E. coli was stored in sealed tubes at 4 and 20°C and the number of viable cells and level of recombinant protein production were analyzed weekly. Different tests showed that the biochemical characteristics of immobilized E. coli remained intact. At both temperatures selected, we found that the number of bacteria in silicon dioxide-derived matrix was of the same order of magnitude (10⁹ cfu ml⁻¹) as before immobilization, for 2 months. After 2 weeks, cells immobilized in an alkoxide-derived matrix decreased to 10⁴ cfu ml⁻¹ at 4°C, and no viable cells were detected at 20°C. We found that immobilized bacteria could be used as a starter to produce recombinant proteins with yields comparable to those obtained from glycerol stocks: 15 mg l⁻¹ for superantigens and 2 mg l⁻¹ for T-cell receptor β chain. These results contribute to the development of methods for microbial cell preservation under field conditions. Martín F. Desimone and Mauricio C. De Marzi contributed equally to this work     

RNAIII-inhibiting peptide improves efficacy of clinically used antibiotics in a murine model of staphylococcal sepsis

RNAIII-inhibiting peptide (RIP, YSPWTNF-NH2) is a quorum-sensing peptide inhibitor that prevents Staphylococcus aureus toxin production and biofilm formation. A mouse sepsis model was used to test the efficacy of RIP alone or in combination with conventional antibiotics in suppressing S. aureus-induced sepsis. Mice were injected intravenously with 3.0x10(6)CFU of S. aureus ATCC 25923 or with 3.0x10(6)CFU of S. aureus strain Smith diffuse. All animals were randomized to receive intravenously isotonic sodium chloride solution as a control, or 20 mg/kg RIP alone or combined with 20 mg/kg cefazolin, 10 mg/kg imipenem, or 10 mg/kg vancomycin immediately or 6 h after bacterial challenge. Main outcome measures were bacteremia and lethality. All compounds reduced lethality when compared to controls. Although, in general combined-treated groups had significant lower bacterial counts when associated to singly-treated groups only the combination between RIP and vancomycin with respect to cefazolin gave a statistically significant decrease in the lethality rate. Lowest lethality rates (10%) and bacteremia (<10(2)CFU/ml) were obtained when RIP was administered in combination with vancomycin. Because RIP can be synergistic with current antibiotic therapies and help to reduce S. aureus exotoxins production, it can be considered a promising agent to associate with antibiotics for further clinical research into treatment of sepsis.     

Prediction of gas-liquid mass transfer coefficient in sparged stirred tank bioreactors

Oxygen mass transfer in sparged stirred tank bioreactors has been studied. The rate of oxygen mass transfer into a culture in a bioreactor is affected by operational conditions and geometrical parameters as well as the physicochemical properties of the medium (nutrients, substances excreted by the micro-organism, and surface active agents that are often added to the medium) and the presence of the micro-organism. Thus, oxygen mass transfer coefficient values in fermentation broths often differ substantially from values estimated for simple aqueous solutions. The influence of liquid phase physicochemical properties on kLa must be divided into the influence on k(L) and a, because they are affected in different ways. The presence of micro-organisms (cells, bacteria, or yeasts) can affect the mass transfer rate, and thus kLa values, due to the consumption of oxygen for both cell growth and metabolite production. In this work, theoretical equations for kLa prediction, developed for sparged and stirred tanks, taking into account the possible oxygen mass transfer enhancement due to the consumption by biochemical reactions, are proposed. The estimation of kLa is carried out taking into account a strong increase of viscosity broth, changes in surface tension and different oxygen uptake rates (OURs), and the biological enhancement factor, E, is also estimated. These different operational conditions and changes in several variables are performed using different systems and cultures (xanthan aqueous solutions, xanthan production cultures by Xanthomonas campestris, sophorolipids production by Candida bombicola, etc.). Experimental and theoretical results are presented and compared, with very good results.     

Partitioning of CO2 Incorporation Among Planktonic Microbial Guilds and Estimation of In Situ Specific Growth Rates

Partitioning of CO₂ incorporation into oxygenic phototrophic, anoxygenic phototrophic, and chemolithoautotrophic guilds was determined in a freshwater lake (Lake Cisó, Banyoles, Spain). CO₂ incorporation into the different types of microorganisms was studied at different depths, during diel cycles, and throughout the year. During winter holomixis, the whole lake became anoxic and both the anoxygenic and chemolithoautotrophic guilds were more active at the surface of the lake, whereas the activity of the oxygenic guild was negligible. During stratification, the latter guild was more active in the upper metalimnion, whereas the anoxygenic guild was more active in the lower metalimnion. Specific growth rates and doubling times were estimated for the most conspicuous phototrophic microorganisms. Doubling times for Cryptomonas phaseolus ranged between 0.5 and 192 days, whereas purple sulfur bacteria (Chromatiaceae-like) ranged between 1.5 and 238 days. These growth rates were similar to those calculated with a different approach in previous papers and indicate slow-growing populations with very large biomass. Overall, the annual total CO₂ incorporation in Lake Cisó was 220 g C m⁻². Most of the CO₂ incorporation, however, was due to the chemolithoautotrophic guild (61% during holomixis and 56% during stratification), followed by the anoxygenic phototrophic guild (35 and 19%, respectively) and the oxygenic phototrophs (4 and 25%, respectively), making dark carbon fixation the key process in the autotrophic metabolism of the lake.     

Perilipin targets a novel pool of lipid droplets for lipolytic attack by hormone-sensitive lipase

Adipocytes serve as the principal energy reservoir of the body; however, the subcellular organization of the machinery regulating lipid trafficking and metabolism is poorly understood. Mobilization of stored triglyceride is thought be controlled by interactions among intracellular lipases and proteins that coat lipid storage droplets. A major limitation of previous studies of hormone-mediated lipolysis, however, is the use of cultured model adipocytes whose three-dimensional architectures do not resemble those in real adipose tissue. To address this limitation, we investigated the intracellular targeting of perilipin, a major lipid coat protein, and hormone-sensitive lipase in three preparations that exhibit more appropriate morphologies: 3T3-L1 adipocytes grown in three-dimensional matrix, dissociated mature adipocytes from mouse adipose tissue, and adipocytes within intact fat pads. High resolution imaging of native and fluorescently tagged proteins indicate that: 1) perilipin preferentially targets a special class of peripheral lipid storage droplets, but not the major or central lipid storage droplets, 2) the peripheral droplets are the sites of attack by hormone-sensitive lipase, and 3) perilipin and hormone-sensitive lipase are continuously colocalized following lipolytic activation. These results indicate that in white adipose tissue, lipolysis takes place in a specialized subcellular domain that is distinct from the major lipid storage site and is defined by perilipin.     

NF-kappaB activation but not PI3K/Akt is required for dexamethasone dependent protection against TNF-alpha cytotoxicity in L929 cells

Tumor necrosis factor alpha (TNF-alpha) is one of the best-described cell death promoters. In murine L929 fibroblasts, dexamethasone inhibits TNF-alpha-induced cytotoxicity. Since phosphatidyl inositol 3 kinase (PI3K) and nuclear factor kappa B (NF-kappaB) proteins regulate several survival pathways, we evaluated their participation in dexamethasone protection against TNF-alpha cell death. We interfered with these pathways by overexpressing a negative dominant mutant of PI3K or a non-degradable mutant of inhibitor of NF-kappaB alpha (IkappaBalpha) (the cytoplasmic inhibitor of NF-kappaB) in L929 cells. The mutant IkappaB, but not the mutant PI3K, abrogated dexamethasone-mediated protection. The loss of dexamethasone protection was associated with a diminished accumulation in XIAP and c-IAP proteins.